EP2118310B1 - Systeme und verfahren zum nachweis von nukleinsäuren - Google Patents

Systeme und verfahren zum nachweis von nukleinsäuren Download PDF

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EP2118310B1
EP2118310B1 EP07871745A EP07871745A EP2118310B1 EP 2118310 B1 EP2118310 B1 EP 2118310B1 EP 07871745 A EP07871745 A EP 07871745A EP 07871745 A EP07871745 A EP 07871745A EP 2118310 B1 EP2118310 B1 EP 2118310B1
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Prior art keywords
probe
region
capture
nucleic acid
solid support
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French (fr)
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EP2118310A1 (de
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Kristian Scaboo
Vissarion Aivazachvili
Timothy Liu
Robert Eason
Konrad Faulstich
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Applied Biosystems LLC
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Applied Biosystems LLC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • This application relates generally to systems and methods for detecting biological molecules and, in particular, to systems and methods for detecting nucleic acids in a sample.
  • Nucleic acid amplification may be performed in conjunction with a variety of assays. Such assays may be qualitative, for example when used to evaluate a biological sample. However, a wide variety of biological applications could be improved by the ability to detect the amplification of target nucleic acids, without requiring either cumbersome blotting techniques, or the expensive and delicate equipment typically required for optical methods.
  • a method of detecting a target nucleic acid in a sample which comprises:
  • a method for detecting a target nucleic in a sample which comprises:
  • a kit for detecting a target nucleic acid in a sample which comprises:
  • capture probe refers to a nucleobase polymer that is surface bound.
  • the capture probe can be a nucleic acid (e.g. DNA or RNA), a nucleic acid analog (e.g. locked nucleic acid (LNA)), a nucleic acid mimic (e.g. peptide nucleic acid (PNA)) or a chimera.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • chimera refers to a nucleobase polymer comprising two or more linked subunits that are selected from different classes of subunits.
  • a PNA/DNA chimera would comprise at least one PNA subunit linked to at least one 2'-deoxyribonucleic acid subunit (For exemplary methods and compositions related to PNA/DNA chimera preparation See: WO96/40709 ).
  • Exemplary component subunits of a chimera are selected from the group consisting of PNA subunits, naturally occurring amino acid subunits, DNA subunits, RNA subunits, LNA subunits and subunits of other analogues or mimics of nucleic acids.
  • overlap refers to a portion of a hybridization probe that is non-complementary to the target nucleic acid the probe is designed to determine.
  • hybridization probe is a nucleobase polymer that can be cleaved by nuclease activity of an enzyme at a site where the probe is hybridized to a complementary strand, said hybridization probe comprising a nucleobase sequence that is complementary to at least a portion of a target nucleic acid of interest in a sample.
  • the hybridization probe can be a oligonucleotide, oligonucleotide analog or chimera so long as it is cleavable by nuclease activity.
  • the nucleobase polymer can be a chimera that comprises all DNA subunits except for one LNA subunit.
  • the nucleobase polymer comprises a single LNA subunit that is situated one subunit removed (toward the 3' end) from the 5' end of that portion of the hybridization probe that is designed to hybridize to the target nucleic acid.
  • nuclease activity refers to the ability of an enzyme to cleave the backbone of a nucleobase polymer (e.g., a nucleic acid).
  • nuclease activity include exonuclease activity (i.e., the ability of an enzyme to cleave nucleotide sequences sequentially from the free end of a nucleobase polymer substrate) and endonuclease activity (i.e., the ability of a protein to recognize specific, short sequences of a nucleobase polymer and to cleave the nucleobase polymer at those sites).
  • nucleobase polymer refers to a polymer comprising a series of linked nucleobase containing subunits.
  • suitable polymers include oligodeoxynucleotides, oligoribonucleotides, peptide nucleic acids, nucleic acid analogs, nucleic acid mimics and chimeras.
  • peptide nucleic acid or “PNA” refers to any polynucleobase strand or segment of a polynucleobase strand comprising two or more PNA subunits, including, but not limited to, any polynucleobase strand or segment of a polynucleobase strand referred to or claimed as a peptide nucleic acid in United States Patent Nos.
  • PNA is a nucleic acid mimic and not a nucleic acid or nucleic acid analog. PNA is not a nucleic acid since it is not formed from nucleotides.
  • PNA oligomers may include polymers that comprise one or more amino acid side chains linked to the backbone.
  • support refers to any solid phase material.
  • Solid support encompasses terms such as “resin”, “synthesis support”, “solid phase”, “surface” “membrane” and/or “support”.
  • a solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof.
  • a solid support can also be inorganic, such as glass, silica, controlled-pore-glass (CPG), or reverse-phase silica.
  • the configuration of a solid support can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface.
  • Solid supports can be porous or non-porous, and can have swelling or non-swelling characteristics.
  • a solid support can be configured in the form of a well, depression, tube, channel, cylinder or other container, vessel, feature or location.
  • target nucleic acid refers to a nucleic acid molecule of interest.
  • a sample can comprise more than one target nucleic acid molecule.
  • a United States patent application with publication number US 2005/0255512 discloses a method of detecting or measuring a target nucleic acid in a sample.
  • the method includes forming a cleavage structure by incubating the sample with a probe.
  • the probe has a binding moiety secondary and a structure that changes upon binding to the target nucleic acid.
  • the method includes cleaving the cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal and thus indicating the presence of the target nucleic acid.
  • the method further includes detecting or measuring the amount of the fragment captured by binding of the binding moiety to a capture element on a solid support.
  • An international application with publication number WO 2001/06016 discloses a method for detecting a target nucleic acid sequence.
  • the method includes hybridizing a primer to the target nucleic acid sequence to form a hybridization complex and contacting the complex with an enzyme to form a modified primer nucleic acid.
  • the complex is disassociated and an assay complex is formed with at least one electron transfer moiety and the modified primer nucleic acid.
  • the assay complex is covalently attached to an electrode and electron transfer between the electron transfer moiety and the electrode is then detected as an indication of the presence of the target nucleic acid sequence.
  • An international application with publication number WO 2005/010199 discloses a further method for detecting target nucleotide sequences.
  • the method uses at least one invasive cleavage reaction to generate tagged molecules having identifier tags corresponding to target nucleotide sequences.
  • the hybridization of any tagged molecule with a complementary detection probe on a universal detector then indicates the presence of the corresponding target in the sample being assayed.
  • the present inventors have discovered that the ability to discriminate between cleaved and intact hybridization probes can also be achieved without separation by modifying the structure of the solid phase capture probe and/or the hybridization probe.
  • the structure of the solid phase capture probe and/or the hybridization probe has an affect on the ability of the capture probe to discriminate between the intact hybridization probe and the cleaved probe fragment. While not wishing to be bound by theory, it is believed that this phenomenon results from a steric hindrance effect that inhibits hybridization of the intact or uncleaved (i.e., the longer or more bulky) hybridization probe to the capture probe.
  • the hybridization probe is substantially single stranded at the T m of the probe fragment/capture probe complex wherein "substantially single stranded" means that less than 5% of the hybridization probe is part of a double stranded complex (e.g., a folded structure).
  • Two electrode capture probe sequences were investigated for their ability to discriminate between the cleaved hybridization probe fragment and the intact hybridization probe.
  • the capture probe sequences employed in the following experiments differ by the distance of nineteen bases between the hybridization region, which is shown in boldface and underlined below, and the gold surface.
  • the sequences of the two capture probes are shown below, with the portion of the sequence homologous to the second region of the probe fragment(s) produced by cleavage of the hybridization probe shown in bold and underlined.
  • the oligonucleotides were modified on the 5' end with the dithiol phosphoramidite (DTPA) (Glen Research, Inc) for attachment to the gold electrodes.
  • the capture probes were attached to the electrodes using the following procedure. First, the electrodes were cleaned by exposure to an UV Ozone Cleaner (Jelight Inc) for 20 min followed by an ethanol soak to reduce the oxide formed. Then, 40 ⁇ L of a 1 uM solution of the thiolated capture probe in 1M Phosphate buffer (pH 7) was deposited on the surface for 15 min in an electrode area defined by a silicone well (Molecular Probes, Inc). The electrodes were then rinsed in water and exposed to a 2 mM mercaptohexanol solution for 2 hrs. After exposure, the electrodes were rinsed in water and dried under argon.
  • DTPA dithiol phosphoramidite
  • the PCR hybridization probe was obtained from IDT Inc. with a 5' amine modification so that it could be coupled in-house to an electroactive Ferrocene (Fc) moiety.
  • the sequence is as follows with the 5' flap indicated in bold and underlined:
  • PCR was performed in 1X buffer A from core PCR kit (Applied Biosystems Ca# N808-0228) supplemented with 6 mM MgCl 2 .
  • PCR primers and Ferrocene labeled hybridization probe were present at concentrations 200 nM and 400 nM, respectively.
  • the 25 ⁇ L, reaction mix contained either 3000 or 0 copies of Listeria DNA for positive and negative (i.e., no template control) samples. Cycling parameters were as follows: 95° C for 10 min., then (95° C for 15 sec and 66° C for 30 sec) X 40 cycles.
  • the 25 ⁇ L reaction mix was placed on the gold electrode and covered with a glass coverslip for I hr static hybridization at room temperature. Alternatively, the PCR mix was introduced into the multiplexed electrode chip for flow-through detection.
  • FIG. 1 is a photograph of a multiplexing chip which can be used for electrochemical measurements.
  • the chip includes 500 ⁇ m wide gold finger working electrodes inter-digitated with a pronged counter electrode crossing a flow channel of 350 ⁇ m width and 120 ⁇ m height.
  • the gold electrodes are modified before assembly with the indicated capture probes and then the chip is assembled. Subsequently, 20 ⁇ L of the completed PCR solutions are flowed through the chip at a flow rate of 1 ⁇ L/ min to allow for hybridization.
  • FIG. 2 shows the results for the planar gold electrode with a static, I hour hybridization.
  • discrimination between the positive sample and the no template control (NTC) can be seen for both capture probes.
  • NTC no template control
  • Capture Probe 2 there is much lower signal from the NTC sample. This indicates that the intact hybridization probe does not hybridize well to that probe surface.
  • the "discrimination ratio" of the capture probe is the ratio obtained by dividing the intensity of the signal generated from the positive sample by the intensity of the signal generated by the no template control (NTC) under the conditions and using the protocol set forth above.
  • FIGS. 4A and 4B illustrate the shorter capture probe which has a hybridization region closer to the surface of the electrode prevents efficient hybridization of the full length, intact hybridization probe to the capture probe.
  • FIGS. 4A and 4B illustrate the hybridization of cleaved and intact hybridization probes to the longer Capture Probe 1
  • FIG. 4B illustrates the hybridization of cleaved and intact hybridization probes to the shorter Capture Probe 2.
  • the ability of the capture probe to discriminate between the probe fragment and the intact hybridization probe can also be enhanced by variations in said hybridization probe.
  • the present inventors have discovered that the length of the 5' flap of the hybridization probe (which 5' flap is part of the probe fragment after cleavage of the hybridization probe by the nuclease activity of the enzyme) also influences the ability of the capture probe to discriminate between cleaved and intact hybridization probe.
  • the probe of SEQ ID NO: 4 When cleaved by the nuclease activity of the enzyme, the probe of SEQ ID NO: 4 produces, when cleaved, a probe fragment comprising the 19 mer 5' flap whereas the probe of SEQ ID NO: 5 produces a probe fragment comprising the 15 mer 5' flap and the probe of SEQ ID NO: 6 produces a probe fragment comprising the 13 mer 5' flap.
  • Bar graphs showing the results for hybridization to a 20 mer capture probe are provided in FIGS. 5A-5C for the each of the probe fragments comprising the 19 mer, 15 mer and 13 mer 5' flaps, respectively.
  • the probe fragment comprising the 13 mer flap produces much higher discrimination between the cleaved and intact hybridization probe than do the probe fragments comprising longer flaps. Thus, it seems that shortening of the 5' flap decreases the binding of the intact hybridization probe to the surface bound capture probe.
  • hybridizations were carried out at temperatures 10° C below the predicted T m of the duplexes formed by the second region (i.e. the nucleobase sequence of the 5' flap) of the probe fragments and the capture probe.
  • FIG. 6 is a schematic depiction of an electrochemical cell having a gold working electrode (WE) and a platinum counter electrode (CE) that can be used in the above described assays.
  • the electrochemical cell is formed by sandwiching a PDMS gasket between the counter and working electrodes.
  • the working electrode (WE) and the counter electrode (CE) can have diameters of 2 mm.
  • the platimun counter-electrode (CE) can be made by sputter coating a 2000 Angstrom thick platinum layer on a silicon wafer having a Cr adhesion layer.
  • the gold counter-electrode (CE) can be made by sputter coating a 2000 Angstrom thick gold layer on a silicon wafer having a Cr adhesion layer.
  • the reference electrode can be a 0.5 mm diameter Ag/AgCl wire.
  • a tag complement is immobilized on an electrode by thiol moieties (here provided by DTPA moieties) that exhibit specificity for binding to gold surfaces, such as a gold electrode, and a cleavable probe that contains (i) a polynucleotide sequence attached to the 5' end of a target complementary segment and (ii) a detectable tag comprising an osmium-containing complex for electrochemical detection after capture of the cleaved tag by the immobilized tag complement.
  • thiol moieties here provided by DTPA moieties
  • a detectable tag comprising an osmium-containing complex for electrochemical detection after capture of the cleaved tag by the immobilized tag complement.
  • the cleaved probe can be detected and/or measured in the presence of uncleaved probe by selection of an appropriate capture probe (a tag complement) such that the capture probe destabilizes capture of uncleaved (intact) probe by selectively binding the tag of the uncleaved probe close to the electrode surface.
  • an appropriate capture probe a tag complement
  • the capture probe hybridizes to the cleaved tag more stably than the uncleaved tag moiety bound to the probe.
  • a 50 ⁇ l reaction mix is prepared that contains 1X PCR buffer A (Applied Biosystems, P/N N808-0228), 6 mM MgCl 2 , 200 ⁇ M of each dNTP, 200 nM of forward and reverse primers, 400 nM 5'-Os-labeled probe, 0.05 units of Gold AmpliTaqTM polymerase and 3,000 copies of Listeria monocytogenesis DNA.
  • 1X PCR buffer A Applied Biosystems, P/N N808-0228
  • 6 mM MgCl 2 200 ⁇ M of each dNTP
  • 200 nM of forward and reverse primers 200 nM of forward and reverse primers
  • 400 nM 5'-Os-labeled probe 0.05 units of Gold AmpliTaqTM polymerase and 3,000 copies of Listeria monocytogenesis DNA.
  • the osmium complex labeling agent that was coupled to the 5' amino group of each probe to form the Os-labeled probe is shown in FIG. 9 along with a scheme for the synthesis of the osmium complexing agent.
  • the forward and reverse primers used during the PCR were as follows:
  • Thermocycling was performed at 95°C for 10 min., then (92° C for 15 sec, 66° C for 30 sec.) X 40 cycles. Then, the PCR mix was loaded into an electrochemical cell of the type depicted in FIG. 6 for electrochemical measurements. The measurements were performed using a 1 M NaCl hybridization buffer at 31°C (which is approximately 10 degrees below the melt temperature (T m ) of the 15-mer cleaved tag sequence in Combination #3 above as calculated using the T m calculator program on IDT web site: www.idt.com). Results are shown in FIG. 7 .
  • FIG. 7 is a bar graph showing the results for hybridization of the three different hybridization probe/probe fragment combinations set forth above.
  • a schematic depiction of the binding of the intact hybridization probes to the capture probe for each of the combinations is shown in FIGS 8A, 8B and 8C .
  • each hybridization probe had a 23 mer region which did not hybridize to the capture probe.
  • the hybridization probes used in Combinations 1 and 2 also had a 19 mer region (i.e. a 5 'flap) that hybridized to the capture probe whereas the hybridization probe used in Combination 3 had a shorter 15 mer region (i.e. 5' flap) that hybridized to the capture probe.
  • the capture probe used in Combination 1 had a 25 mer spacer region between the support surface and the region that hybridizes to the hybridization probe.
  • the capture probes used in Combinations 2 and 3 did not have the 25 mer spacer region between the support surface and the region that hybridizes to the 5' flap of the hybridization probe.
  • Combination 3 provided by far the highest level of discrimination between the cleaved hybridization probe fragment and the intact hybridization probe.
  • the hybridization probe used in Combination 3 had the shortest region which hybridized to the capture probe (15 mers). For Combinations 1 and 2, hybridization to the capture probe was conducted at 42° C whereas for Combination 3, hybridization was conducted to 32° C.
  • the ability of the capture probe to discriminate between the probe fragment and the intact hybridization probe can be enhanced by variations in both the sequence and length of the capture probe as well as by modifications of the hybridization probe.
  • the hybridization probe can also be modified by extending the 3' end of the hybridization probe or by adding a bulky modification on the 3' end of the hybridization probe that would further block access to the capture probe sequence on the solid support surface.
  • electrochemical label can be any known electrochemical moiety as a label on the cleaved portion of the hybridization probe.
  • Exemplary electrochemical labels which may be used include bis(2,2'-bipyridyl)imidizolylchloroosmium(II) [salt]. This label gives a good E o of 0.165 vs Ag/AgCl and has good solubility properties for synthesis and purification.
  • Other exemplary labels include ferrocene as well as the labels disclosed in U.S. Patent Application No. 11/488,439 filed on July 17, 2006 .
  • the electrochemical label can be any moiety that can transfer electrons to or from an electrode.
  • Exemplary electrochemical labels include transition metal complexes.
  • Suitable transition metal complexes include, for example, ruthenium 2+ (2,2'-bipyridine) 3 (Ru(bpy) 3 2+ ), ruthenium 2+ (4,4'-dimethyl-2,2'-bipyridine) 3 (Ru(Me 2 -bpy) 3 2+ ), ruthenium 2+ (5,6-dimethyl-1,10-phenanthroline) 3 (Ru(Me 2 -phen) 3 2+ ), iron 2+ (2,2'-bipyridine) 3 (Fe(bpy) 3 2+ ), iron 2+ (5-chlorophenanthroline) 3 (Fe(5-Cl-phen) 3 2+ ), osmium 2+ (5-chlorophenanthroline) 3 (Os(5-Cl-phen) 3 2+ ), osmium 2+ (2,2'-bipyridine) 2 (imidazolyl), dioxorhenium 1+ phosphine, and dio
  • Some anionic complexes useful as mediators are: Ru(bpy)((SO 3 ) 2 -bpy) 2 2- and Ru(bpy)((CO 2 ) 2 -bpy) 2 2- and some zwitterionic complexes useful as mediators are Ru(bpy) 2 ((SO 3 ) 2 -bpy) and Ru(bpy) 2 ((CO 2 ) 2 -bpy) where (SO 3 ) 2 -bpy 2 - is 4,4'-disulfonato-2,2'-bipyridine and (CO 2 ) 2 -bpy 2 - is 4,4'-dicarboxy-2,2'-bipyridine.
  • Suitable substituted derivatives of the pyridine, bypyridine and phenanthroline groups may also be employed in complexes with any of the foregoing metals.
  • Suitable substituted derivatives include but are not limited to 4-aminopyridine, 4-dimethylpyridine, 4-acetylpyridine, 4-nitropyridine, 4,4'-diamino-2,2'-bipyridine, 5,5'-diamino-2,2'-bipyridine, 6,6'-diamino-2,2'-bipyridine, 4,4'-diethylenediamine-2,2'-bipyridine, 5,5'-diethylenediamine-2,2'-bipyridine, 6,6'-diethylenediamine-2,2'-bipyridine, 4,4'-dihydroxyl-2,2'-bipyridine, 5,5'-dihydroxyl-2,2'-bipyridine, 6,6'-dihydroxyl-2,2'-bipyridine, 4,4
  • the disclosed methods are also applicable to the detection of nucleic acids by other detection techniques, such as fluorescence detection.
  • the detectable label on the hybridization probe can be any moiety which is capable of being detected and/or quantitated.
  • Exemplary labels include electrochemical, luminescent (e.g., fluorescent, luminescent, or chemiluminescent) and colorimetric labels.
  • the primers and probes used herein may have any of a variety of lengths and configurations.
  • the primers may be from 18 to about 30 subunits in length or from 20 to 25 subunits in length. Longer or shorter length primers can also be used.
  • the length of the region of the hybridization probe which binds to the target nucleic acid can be from 8 to 30 subunits whereas the length of the region of the hybridization probe which does not bind to the target can have a length of 2 to 40 subunits or from 8 to 30 subunits.
  • Hybridization probes having longer or shorter regions than those exemplified above can also be used.
  • the primers may be designed to bind to and produce an amplified product of any desired length, usually at least 30 or at least 50 nucleotides in length and up to 200, 300, 500, 1000, or more nucleotides in length.
  • the probes and primers may be provided at any suitable concentrations.
  • forward and reverse primers for PCR may be provided at concentrations typically less than or equal to 500 nM, such as from 20 nM to 500 nm, or 50 to 500 nM, or from 100 to 500 nM, or from 50 to 200 nM.
  • Probes are typically provided at concentrations of less than or equal to 1000 nM, such as from 20 nM to 500 nm, or 50 to 500 nM, or from 100 to 500 nM, or from 50 to 200 nM. Exemplary conditions for concentrations of NTPs, enzyme, primers and probes can also be found in U.S. Patent No. 5,538,848 or can be achieved using commercially available reaction components (e.g., as can be obtained from Applied Biosystems, Foster City, CA).
  • a plurality of complementary capture probes may also be used.
  • an array of capture oligonucleotides that hybridize to different hybridization probe fragments may be used to localize and capture individual tag sequences in a plurality of discrete detection zones.
  • the methods described herein can be used to detect target nucleic acid in real time.
  • the solid support can be in contact with the solution in which nucleic acid amplification is occurring and the process monitored during PCR (i.e. real-time detection).
  • detection of probe fragments can also be conducted after the amplification process is complete (i.e., end-point detection).
  • the PCR assay can be monitored during PCR (real-time) and after the process is completed (i.e. end-point).
  • PCR assays can be performed using traditional PCR formats as well as Fast PCR formats, asymmetric PCR formats and asynchronous PCR formats.

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Claims (22)

  1. Verfahren zum Nachweisen einer Zielnukleinsäure in einer Probe, wobei das Verfahren umfasst:
    (a) Inkubieren der Probe mit:
    (1) einem Primer, der mit zumindest einem Abschnitt der Zielnukleinsäure hybridisiert;
    (2) eine Hybridisierungssonde, die erste und zweite Regionen umfasst, wobei die erste Region mit zumindest einem Abschnitt der Zielnukleinsäure hybridisiert, und wobei die zweite Region nicht mit der Zielnukleinsäure hybridisiert, wobei die zweite Region eine nachweisbare Markierung umfasst; und
    (3) eine Polymerase und ein Enzym mit Nukleaseaktivität, wobei die Polymerase den hybridisierten Primer in Richtung der hybridisierten Sonde erweitert und die Nukleaseaktivität des Enzyms die hybridisierte Sonde spaltet, um ein Sondenfragment freizusetzen, das die zweite Region und die nachweisbare Markierung umfasst;
    (b) Ermöglichen, dass der Primer und die Hybridisierungssonde mit der Zielnukleinsäure in der Probe hybridisieren;
    (c) Ermöglichen, dass die Polymerase den hybridisierten Primer erweitert;
    (d) Ermöglichen, dass die Nukleaseaktivität des Enzyms die hybridisierte Hybridisierungssonde spaltet, um das Sondenfragment freizusetzen;
    (e) Inkontaktbringen der Probe mit einer Oberfläche eines Feststoffträgers, wobei die Oberfläche des Feststoffträgers eine oder mehrere Fängersonden umfasst, wobei jede davon mit zumindest einem Abschnitt der zweiten Region des Sondenfragments hybridisiert;
    (f) Ermöglichen, dass die Fängersonden mit dem Sondenfragment in der Probe hybridisieren, um einen Sondenfragment/Fängersonde-Komplex zu bilden; und
    (g) Nachweisen der Markierung auf der Oberfläche des Feststoffträgers,
    wobei die Hybridisierungssonde bei der Tm des Sondenfragment/Fängersonde-Komplexes im Wesentlichen einzelsträngig ist, und wobei die erste Region der Hybridisierungssonde eine Einheit umfasst, die die Bindung der intakten Hybridisierungssonde an die Fängersonde durch sterische Hinderung hemmt.
  2. Verfahren nach Anspruch 1, wobei die zweite Region des Sondenfragments an die Fängersonde bindet, so dass der Abschnitt der zweiten Region benachbart der ersten Region in der intakten Hybridisierungssonde hin zur Oberfläche des Feststoffträgers ausgerichtet ist, wobei optional eine proximale Region der Fängersonde benachbart der Oberfläche des Feststoffträgers nicht mit dem Sondenfragment hybridisiert und eine distale Region der Fängersonde weg von der Oberfläche des Feststoffträgers mit der zweiten Region des Sondenfragments hybridisiert.
  3. Verfahren nach Anspruch 2, wobei die proximale Region der Fängersonde kürzer als die erste Region der Hybridisierungssonde ist.
  4. Verfahren nach Anspruch 1, wobei die Fängersonde und die Hybridisierungssonde jeweils Polynukleotide umfassen, wobei die Polynukleotide optional Desoxyribonukleotide umfassen.
  5. Verfahren nach Anspruch 4, wobei die proximale Region der Fängersonde weniger Nukleotide als die erste Region der intakten Hybridisierungssonde aufweist.
  6. Verfahren nach Anspruch 1, wobei die Hybridisierungssonde ferner eine dritte Region benachbart der ersten Region und gegenüber der zweiten Region umfasst, wobei die dritte Region nicht mit der Zielnukleinsäure hybridisiert.
  7. Verfahren nach Anspruch 1, umfassend:
    (a) Schmelzen der Probe durch Erhitzen der Probe auf eine erste Temperatur, wobei die erste Temperatur über der Schmelztemperatur (Tm) des Primers liegt, und wobei doppelsträngige Nukleinsäuren in der Probe vorhanden sind, die die Zielnukleinsäure umfassen;
    (b) danach Annelieren der Probe durch Verringern der Temperatur auf eine zweite Temperatur, die niedriger als die erste Temperatur ist, um es dem Primer und der Hybridisierungssonde zu ermöglichen, jeweils mit einem einzelsträngigen Abschnitt der Zielnukleinsäure in der Probe zu hybridisieren; und
    (c) danach Verlängern des Primers, indem es der Polymerase ermöglicht wird, den mit der Zielnukleinsäure hybridisierten Primer bei einer dritten Temperatur zu erweitern, wodurch das Sondenfragment freigesetzt wird;
    (d) optional zumindest einmaliges Wiederholen des Schmelzens, Annelierens und Verlängerns;
    (e) Inkontaktbringen der Probe mit einer Oberfläche eines Feststoffträgers, wobei die Oberfläche des Feststoffträgers eine oder mehrere Fängersonden umfasst, die mit zumindest einem Abschnitt der zweiten Region des Sondenfragments hybridisieren;
    (f) Ermöglichen, dass die Fängersonden mit zumindest einem Abschnitt des Sondenfragments in der Probe hybridisieren, um einen Sondenfragment/Fängersonde-Komplex bei einer vierten Temperatur zu bilden, die niedriger als die zweite und die dritte Temperatur ist; und
    (g) Nachweisen der Markierung auf der Oberfläche des Feststoffträgers.
  8. Verfahren nach einem vorstehenden Anspruch, wobei die Fängersonde ein Diskrimierungsverhältnis von 3 oder höher aufweist.
  9. Verfahren nach Anspruch 7, wobei die zweite Temperatur und die dritte Temperatur gleich sind.
  10. Verfahren nach einem vorstehenden Anspruch, wobei die Polymerase und das Enzym mit Nukleaseaktivität das gleiche Molekül sind, wobei die Polymerase optional ein wärmestabiles Enzym ist.
  11. Verfahren nach einem vorstehenden Anspruch, wobei die Oberfläche des Feststoffträgers eine Elektrode umfasst, und wobei die nachweisbare Markierung eine Einheit ist, die Elektronen von der oder zur Elektrode übertragen kann, wobei die nachweisbare Markierung optional eine Ferroceneinheit ist, und/oder wobei die Oberfläche des Feststoffträgers optional Gold umfasst.
  12. Verfahren nach einem vorstehenden Anspruch, wobei der Feststoffträger eine Vielzahl von fingerförmig ineinandergreifenden Platten umfasst, die einen Strömungskanal bilden, wobei zumindest einige der Oberflächen der Platten Fängersonden umfassen, und wobei das Inkontaktbringen der Probe mit einer Oberfläche eines Feststoffträgers das Strömen der Probe durch den Strömungskanal umfasst, wobei die Oberflächen der Platten optional Elektroden umfassen, und/oder wobei die Oberflächen von alternierenden Platten optional Fängersonden umfassen.
  13. Verfahren nach Anspruch 7, wobei das Schmelzen, das Annelieren und das Verlängern mehrmals in einer Reihe von Zyklen durchgeführt werden, wobei das Nachweisen der Markierung auf der Oberfläche des Feststoffträgers optional nach dem letzten Schmelz-, Annelier- und Verlängerungszyklus erfolgt.
  14. Verfahren nach Anspruch 7, wobei die Probe während des Schmelzens, Annelierens und Verlängerns mit der Oberfläche des Feststoffträgers in Kontakt steht, und wobei das Nachweisen mehrmals während des Verfahrens und/oder nach dem letzten Schmelz-, Annelier- und Verlängerungszyklus erfolgt.
  15. Kit zum Nachweisen einer Zielnukleinsäure in einer Probe, umfassend:
    (a) eine Hybridisierungssonde mit einer ersten Region, die mit zumindest einem Abschnitt der Zielnukleinsäure hybridisiert, und mit einer zweiten Region, die eine nachweisbare Markierung umfasst, wobei die zweite Region nicht mit der Zielnukleinsäure hybridisiert, und wobei ein Enzym mit Nukleaseaktivität die Hybridisierungssonde spalten kann, wenn mit der Zielnukleinsäure hybridisiert, um ein Sondenfragment zu produzieren, das die zweite Region und die nachweisbare Markierung umfasst;
    (b) einen Feststoffträger, der eine oder mehrere Fängersonden auf einer Oberfläche davon umfasst, wobei die Fängersonde mit zumindest einem Abschnitt der zweiten Region des Sondenfragments hybridisiert, um einen Sondenfragment/Fängersonde-Komplex zu bilden, wobei die erste Region der Hybridisierungssonde eine Einheit umfasst, die die Bindung der intakten Hybridisierungssonde an die Fängersonde durch sterische Hinderung hemmt, und wobei die Hybridisierungssonde bei der Tm des Sondenfragment/Fängersonde-Komplexes im Wesentlichen einzelsträngig ist;
    (c) optional einen Primer, der mit zumindest einem Abschnitt der Zielnukleinsäure hybridisiert; und
    (d) optional eine Polymerase, die den hybridisierten Primer in Richtung der hybridisierten Sonde erweitert, und ein Enzym mit Nukleaseaktivität, um die hybridisierte Hybridisierungssonde zu spalten und das Sondenfragment, das die zweite Region der Sonde und die nachweisbare Markierung umfasst, freizusetzen.
  16. Kit nach Anspruch 15, wobei die Fängersonde ein Diskrimierungsverhältnis von 3 oder höher aufweist.
  17. Kit nach Anspruch 15, wobei die Oberfläche des Feststoffträgers eine Elektrode umfasst, und wobei die nachweisbare Markierung eine Einheit ist, die Elektronen von der oder zur Elektrode übertragen kann, vorzugsweise wobei die nachweisbare Markierung optional eine elektroaktive Ferroceneinheit ist; und/oder wobei die Oberfläche des Feststoffträgers optional Gold umfasst.
  18. Kit nach Anspruch 15, wobei die Polymerase und das Enzym mit Nukleaseaktivität das gleiche Molekül sind.
  19. Verfahren nach einem der Ansprüche 1 bis 14 oder Kit nach einem der Ansprüche 15 bis 18, wobei die Nukleaseaktivität Exonukleaseaktivität ist.
  20. Verfahren nach Anspruch 8, wobei die Fängersonde ein Diskrimierungsverhältnis von 5 oder höher aufweist.
  21. Verfahren nach Anspruch 10, wobei die Polymerase eine Taq-Polymerase ist.
  22. Kit nach Anspruch 16, wobei die Fängersonde ein Diskrimierungsverhältnis von 5 oder höher aufweist.
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PCT/US2007/089010 WO2008083261A1 (en) 2006-12-29 2007-12-28 Systems and methods for detecting nucleic acids

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8703653B2 (en) 2011-02-18 2014-04-22 NVS Technologies, Inc. Quantitative, highly multiplexed detection of nucleic acids

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012096430A1 (en) 2011-01-11 2012-07-19 Seegene, Inc. Detection of target nucleic acid sequences by pto cleavage and extension assay
CA2827040A1 (en) * 2011-02-18 2012-08-23 NVS Technologies, Inc. Quantitative, highly multiplexed detection of nucleic acids
US11078525B2 (en) 2011-03-29 2021-08-03 Seegene, Inc. Detection of target nucleic acid sequence by PTO cleavage and extension-dependent cleavage
US9850524B2 (en) 2011-05-04 2017-12-26 Seegene, Inc. Detection of target nucleic acid sequences by PO cleavage and hybridization
MX342067B (es) * 2011-05-04 2016-09-09 Seegene Inc Detección de secuencias de ácido nucleico objetivo por desdoblamiento e hibridización de oligonucleótido de sonda.
KR20160100415A (ko) 2011-09-29 2016-08-23 루미넥스 코포레이션 가수분해 프로브
KR20140094007A (ko) * 2011-11-17 2014-07-29 엔브이에스 테크놀로지스 인코포레이티드 핵산의 고도로 다중화된 정량 검출
KR20130101952A (ko) 2012-02-02 2013-09-16 주식회사 씨젠 Ｐｔｏ 절단과 연장-의존적 혼성화를 이용한 타겟 핵산서열의 검출
ES2669244T3 (es) 2012-03-05 2018-05-24 Seegene, Inc. Detección de variación de nucleótidos en una secuencia de ácido nucleico diana mediante escisión CTO y ensayo de extensión
US9683259B2 (en) 2012-04-19 2017-06-20 Seegene, Inc. Detection of target nucleic acid sequence by PTO cleavage and extension-dependent signaling oligonucleotide cleavage
WO2013187628A1 (en) * 2012-06-11 2013-12-19 Seegene, Inc. Detection of target nucleic acid sequence by pto cleavage and extension-dependent transcription
ES2653642T3 (es) 2012-12-27 2018-02-08 Seegene, Inc. Detección de una secuencia de ácido nucleico diana mediante ensayo sin hibridación dependiente de escisión y extensión de PTO
EP2770065B1 (de) 2013-02-25 2017-12-13 Seegene, Inc. Detektion einer Nukleotidvariation auf Nukleinsäurezielsequenz
US20160244816A1 (en) 2013-07-15 2016-08-25 Seegene, Inc. Detection of target nucleic acid sequence by pto cleavage and extension-dependent immobilized oligonucleotide hybridization
EP3058105B1 (de) 2013-10-18 2019-05-22 Seegene, Inc. Nachweis einer zielnukleinsäuresequenz in fester phase durch pto-spaltung und extension mit einem hcto-test
KR101750464B1 (ko) 2014-11-28 2017-06-28 케이맥바이오센터주식회사 바이오물질의 실시간 정량 및 정성 분석을 위한 프로브 시스템, 상기 프로브 시스템을 구비한 반응용기 및 이를 이용한 분석 방법
US20170044596A1 (en) * 2015-07-17 2017-02-16 Luminex Corporation Methods and compositions for catalytic assays
DE102015115836A1 (de) 2015-09-18 2017-03-23 Biotype Diagnostic Gmbh Bestätigungstest für primäre Nukleinsäure-Amplifikate in einem kontinuierlichen Reaktionsansatz und unmittelbare Auswertung mittels immunchromatographischer Verfahren
KR102468174B1 (ko) 2016-09-15 2022-11-17 에프. 호프만-라 로슈 아게 멀티플렉스 pcr 수행 방법
RU2665631C2 (ru) 2017-01-25 2018-09-03 Общество с ограниченной ответственностью "Секвойя Дженетикс" Способ специфической идентификации последовательностей днк
CN108823287B (zh) 2017-04-28 2019-04-12 厦门大学 一种检测靶核酸序列的方法
WO2019066461A2 (en) 2017-09-29 2019-04-04 Seegene, Inc. DETECTION OF TARGET NUCLEIC ACID SEQUENCES BY CLEAVAGE ANALYSIS AND EXTENSION OF PTO
WO2023043280A1 (ko) 2021-09-17 2023-03-23 주식회사 씨젠 합성 비자연 염기를 포함하는 태그 올리고뉴클레오타이드를 이용한 타겟 핵산 서열의 검출

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US5210015A (en) * 1990-08-06 1993-05-11 Hoffman-La Roche Inc. Homogeneous assay system using the nuclease activity of a nucleic acid polymerase
DK51092D0 (da) * 1991-05-24 1992-04-15 Ole Buchardt Oligonucleotid-analoge betegnet pna, monomere synthoner og fremgangsmaade til fremstilling deraf samt anvendelser deraf
US5714331A (en) * 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5641625A (en) * 1992-05-22 1997-06-24 Isis Pharmaceuticals, Inc. Cleaving double-stranded DNA with peptide nucleic acids
US5539082A (en) * 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5719262A (en) * 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5766855A (en) * 1991-05-24 1998-06-16 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity and sequence specificity
US5846717A (en) * 1996-01-24 1998-12-08 Third Wave Technologies, Inc. Detection of nucleic acid sequences by invader-directed cleavage
GB9211979D0 (en) * 1992-06-05 1992-07-15 Buchard Ole Uses of nucleic acid analogues
US5527675A (en) * 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
DE4331012A1 (de) * 1993-09-13 1995-03-16 Bayer Ag Nukleinsäuren-bindende Oligomere mit N-Verzweigung für Therapie und Diagnostik
US5538848A (en) * 1994-11-16 1996-07-23 Applied Biosystems Division, Perkin-Elmer Corp. Method for detecting nucleic acid amplification using self-quenching fluorescence probe
GB2284209A (en) * 1993-11-25 1995-05-31 Ole Buchardt Nucleic acid analogue-induced transcription of RNA from a double-stranded DNA template
US5985557A (en) * 1996-01-24 1999-11-16 Third Wave Technologies, Inc. Invasive cleavage of nucleic acids
US6107470A (en) * 1997-05-29 2000-08-22 Nielsen; Peter E. Histidine-containing peptide nucleic acids
US6290839B1 (en) * 1998-06-23 2001-09-18 Clinical Micro Sensors, Inc. Systems for electrophoretic transport and detection of analytes
WO2000048990A1 (en) * 1999-02-18 2000-08-24 The Regents Of The University Of California Phthalamide-lanthanide complexes for use as luminescent markers
EP1194593A2 (de) * 1999-07-20 2002-04-10 Clinical Micro Sensors, Inc. Verfielfältigung von nukleinsäuren mit elekronischer detektion
US20070134686A1 (en) * 1999-10-29 2007-06-14 Stratagene California Methods and compositions for detection of a target nucleic acid sequence utilizing a probe with a 3' flap
US7118860B2 (en) * 1999-10-29 2006-10-10 Stratagene California Methods for detection of a target nucleic acid by capture
US6350580B1 (en) * 2000-10-11 2002-02-26 Stratagene Methods for detection of a target nucleic acid using a probe comprising secondary structure
AU2002363628A1 (en) * 2001-07-17 2003-05-26 Stratagene Methods for detection of a target nucleic acid by capture using multi-subunit probes
US20030203384A1 (en) * 2002-03-08 2003-10-30 Chafin David R. Multiplex detection of biological materials in a sample
WO2004020654A2 (en) * 2002-08-30 2004-03-11 Bayer Healthcare Llc Solid phase based nucleic acid assays combining high affinity and high specificity
AU2003260292A1 (en) * 2002-09-11 2004-04-30 Exiqon A/S A population of nucleic acids including a subpopulation of lna oligomers
US20070092880A1 (en) * 2003-07-16 2007-04-26 Crothers Donald M Invasive cleavage reaction with electrochemical readout
US7964344B2 (en) * 2003-09-17 2011-06-21 Canon Kabushiki Kaisha Stable hybrid
WO2005068660A1 (en) * 2003-12-19 2005-07-28 Beckman Coulter, Inc. Solid-phase multiplexed invader assay
WO2006079049A2 (en) * 2005-01-21 2006-07-27 Third Wave Technologies, Inc. Methods and compositions for increased dynamic range detection of nucleic acid molecules
EP1907586A2 (de) * 2005-07-15 2008-04-09 Applera Corporation Nukleinsäureamplifikationsnachweis

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8703653B2 (en) 2011-02-18 2014-04-22 NVS Technologies, Inc. Quantitative, highly multiplexed detection of nucleic acids

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